(Not Applicable).
This invention relates to the use of adeno-associated virus (AAV) vectors to lower levels of tumor necrosis factor (TNF). More specifically, the invention relates to AAV vectors encoding a TNF antagonist and methods of using the AAV vectors to reduce the levels of TNF in an individual.
Tumor necrosis factor-xcex1 (TNFxcex1) and tumor necrosis factor-xcex2 (TNFxcex2) are homologous multifunctional cytokines; the great similarities in structural and functional characteristics of which have resulted in their collective description as tumor necrosis factor or xe2x80x9cTNF.xe2x80x9d Activities generally ascribed to TNF include: release of other cytokines including IL-1, IL-6, GM-CSF, and IL-10, induction of chemokines, increase in adhesion molecules, growth of blood vessels, release of tissue destructive enzymes and activation of T cells. See, for example, Feldmann et al., 1997, Adv. Immunol., 64:283-350, Nawroth et al., 1986, J. Exp. Med., 163:1363-1375; Moser et al., 1989, J. Clin. Invest., 83:444-455; Shingu et al., 1993, Clin. Exp. Immunol. 94:145-149; MacNaul et al., 1992, Matrix Suppl., 1:198-199; and Ahmadzadeh et al., 1990, Clin. Exp. Rheumatol. 8:387-391. All of these activities can serve to enhance an inflammatory response.
TNF initiates its biological effect through its interaction with specific, cell surface receptors on TNF-responsive cells. There are two distinct forms of the cell surface tumor necrosis factor receptor (TNFR), designated p75 (or Type II) and p55 (or Type I) (Smith et al., 1990, Science 248:1019-1023; Loetscher et al., 1990, Cell 61:351-359). TNFR Type I and TNFR Type II each bind to both TNFxcex1 and TNFxcex2. Soluble, truncated versions of the TNFRs with a ligand-binding domain are present in body fluids and joints (Engelmann et al., 1989, J. Biol. Chem. 264:11974-11980; Roux-Lombard et al., 1993, Arthritis Rheum. 36:485-489).
A number of disorders are associated with elevated levels of TNF, many of them of significant medical importance. Among such TNF-associated disorders are congestive heart failure, inflammatory bowel diseases (including Crohn""s disease), arthritis and asthma.
TNF appears to effect the heart and endothelium in congestive heart failure and has been implicated in the initiation of an apoptotic process in cardiac myocytes. The role for TNF in this disease is also supported by a temporal association between TNF activation and a transition from asymptomatic to symptomatic congestive heart failure (Ceconi et al., 1998, Prog. Cardiovasc. Dis. 41:25-30).
Inflammatory bowel diseases, such as Crohn""s disease and ulcerative colitis, are associated with increased expression of TNF (Evans et al., 1997, Aliment. Pharmacol. Ther. 11:1031-1035). Treatment of such disorders have included the widespread use of immunosuppressive agents, such as azathioprine, methotrexate, cyclosporine and glucocorticosteroids (Rutgeerts, 1998, Digestion 59:453-469).
Arthritis is a common crippling condition for which there are no cures and few effective therapies. Approximately one in seven people in the United States are affected by one or more forms of arthritis. Most forms of arthritis are characterized by chronic inflammation of joints resulting from infection, mechanical injury, or immunological disturbance. Rheumatoid arthritis (RA) is a chronic inflammatory disease primarily manifest in the joints by swelling, pain, stiffness, and tissue destruction (Harris, 1990, N. Engl. J. Med, 323:994-996). Systemic manifestations can include elevations in serum levels of acute phase proteins, fever, mild anemia, thrombocytosis, and granulocytosis. In affected joints, there is a synovitis characterized by hyperplasia and inflammation of the synovium with an inflammatory exudate into the joint cavity, leading to erosion of cartilage and bone.
Although rheumatoid arthritis is not directly and imminently life threatening, recent data suggest that RA results in significantly shorter lifespan, and puts an enormous toll on the both the health system, the overall economy due to lost productivity, as well as quality of life resulting from restricted mobility and activities (Schiff, 1997, Am. J. Med., 102(1A):11S-15S).
Current commonly employed therapeutics for treatment of RA fall primarily in three categories: non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs), and immunosuppressives. NSAIDs are a large group of drugs often used as first line therapy for rheumatoid arthritis. The compounds act primarily through blockade of cyclooxygenase which catalyzes conversion of arachidonic acid to prostaglandins and thromboxanes. As a class, DMARDs, including agents such as gold, sulfasalazine, hydroxychloroquine, and D-penicillamine, are slow acting, quite toxic and there is little evidence that any of these compounds have mitigating effects on the underlying disease. NSAIDs can relieve some of the signs of inflammation and pain associated with arthritis; however, they appear to be ineffective against the immune system and in blocking progression of joint destruction and disease. Immunosuppressive agents, such as corticosteroids and methotrexate, are commonly used in the treatment of RA for suppressing the immune system and thus having an anti-inflammatory effect. However, these agents engender serious systemic toxicity which limits their use and effectiveness.
Although it is widely accepted that RA is an immune-based inflammatory disease, the antigen(s) which trigger the disease remain unknown. This has led to a large number of approaches to therapy under pre-clinical or clinical investigation which involve attempts to modulate the immune response system as a whole. Examples of several general efforts in this direction are highlighted below.
The mechanism of action of NSAIDs has been linked to blocking of cyclooxygenase, an enzyme with both an inducible and a constitutive form. As the inducible form of cyclooxygenase appears to be elevated in inflammatory disease, investigation into compounds selective for the inducible form are underway. In addition, attempts to devise vaccines to treat ongoing arthritis have been made with the use of peptide vaccines directed toward MHC class II or T cell receptor proteins. Generally, it has been proven difficult to demonstrate efficacy of vaccines administered to ongoing disease.
Much of the tissue destruction in RA appears to be due to various metalloproteinases. This group of proteases are believed to be central to the degradation of collagen II and proteoglycan seen in arthritis. A number of inhibitors of various of these enzymes are under pre-clinical or clinical investigation.
A number of broadly immunosuppressive drugs are in clinical testing for use in rheumatoid arthritis, including cyclosporine A and mycophenolate mofetil. As a wide range of cytokines are found in arthritic joints, anti-arthritis therapies have targeted cytokine pathways including those of IL-1, IL-2, IL-4, IL-10, IL-11, TGFxcex2, and TNFxcex1, as well as, chemokine pathways (Feldmann et al., 1997). In particular, proinflammatory pathways of IL-1 have been targeted both by attack of IL-1 directly and via the naturally occurring interleukin-1 receptor antagonist molecule.
Methods of administering drug therapy for RA have included, and have been proposed to include, systemic or local delivery of a therapeutic drug and, in the case of proposed gene therapies, of a therapeutic gene. To date, such treatments have fallen short of delivering effective, safe therapy for arthritis for a variety of reasons, including: systemic side effects of many drugs, rapid clearance of therapeutic molecules from injected joints and/or circulation, inefficiency in DNA integration and expression from the genome, limited target cell population associated with some viral delivery vectors, transient gene expression associated with viral vectors which do not readily integrate and induction of an immune response associated with the gene delivery virus.
Use of TNF antagonists, such as soluble TNFRs and anti-TNF antibodies, has shown that a blockade of TNF can reverse effects attributed to TNF including decreases in IL-1, GM-CSF, IL-6, IL-8, adhesion molecules and tissue destruction (Feldmann et al., 1997). Such pleiotropic effects apparently due to the blockade of TNF alone suggests that TNF may lie near the top of the cascade of cytokine mediated events. Elevated levels of TNF-xcex1 are found in the synovial fluid of RA patients (Camussi and Lupia, 1998, Drugs 55:613-620).
The effect of TNF blockade utilizing a hamster anti-mouse TNF antibody was tested in a model of collagen type II arthritis in DBA/1 mice (Williams et al., 1992, Proc. Natl. Acad. Sci. USA, 89:9784-9788). Treatment initiated after the onset of disease resulted in improvement in footpad swelling, clinical score, and histopathology of joint destruction. Other studies have obtained similar results using either antibodies (Thorbecke et al., 1992, Proc. Natl. Acad. Sci. USA, 89:7375-7379) or TNFR constructs (Husby et al., 1988, J. Autoimmun. 1:363-71; Tetta et al., 1990, Ann. Rheum. Dis. 49:665-667; Wooley et al., 1993, J. Immunol. 151:6602-6607; Piguet et al., 1992, Immunology 77:510-514).
Similar results have also been obtained in other animal models of ongoing arthritis. In the rabbit, anti-TNFxcex1 antibody was shown to have an anti-arthritic effect on antigen induced arthritis (Lewthwaite et al., 1995, Ann. Rheum. Dis. 54:366-374). In the rat, anti-TNF therapy has been demonstrated to be effective in adjuvant (Mycobacterium) arthritis (Issekutz et al., 1994, Clin. Exp. Immunol. 97:26-32), in streptococcal cell wall induced arthritis (Schimmer et al., 1997, J. Immunol. 159:4103-4108) and in collagen induced arthritis (Le et al., 1997, Arthritis Rheum. 40:1662-1669).
In the studies described above, the TNF blockade was achieved by systemic delivery of the blocking agent. In a rat collagen arthritis model, delivery of a TNFR gene using an adenoviral vector resulted in transient production of serum levels of TNFR (up to 8 days) and a significant decrease in disease progression when the adenovirus was given to animals undergoing active arthritis (Le et al., 1997). Attempts to deliver the gene directly to the joint were unsuccessful, however, and resulted in an inflammatory reaction to the adenovirus.
A monoclonal antibody directed against TNFxcex1 (infliximab, REMICADE, Centocor), administered with and without methotrexate, has demonstrated clinical efficacy in the treatment of RA (Elliott et al., 1993, Arthritis Rheum. 36:1681-1690; Elliott et al., 1994, Lancet 344:1105-1110). These data demonstrate significant reductions in Paulus 20% and 50% criteria at 4, 12 and 26 weeks. This treatment is administered intravenously and the anti-TNF monoclonal antibody disappears from circulation over a period of two months. The duration of efficacy appears to decrease with repeated doses. The patient can generate antibodies against the anti-TNF antibodies which limit the effectiveness and duration of this therapy (Kavanaugh et al., 1998, Rheum. Dis. Clin. North Am. 24:593-614). Administration of methotrexate in combination with infliximab helps prevent the development of anti-infliximab antibodies (Maini et al., 1998, Arthritis Rheum. 41:1552-1563). Infliximab has also demonstrated clinical efficacy in the treatment of the inflammatory bowel disorder Crohn""s disease (Baert et al., 1999, Gastroenterology 116:22-28).
Clinical trials of a recombinant version of the soluble human TNFR (p75) linked to the Fc portion of human IgG1 (sTNFR(p75):Fc, ENBREL, Immunex) have shown that its administration resulted in significant and rapid reductions in RA disease activity (Moreland et al., 1997, N. Eng. J. Med., 337:141-147). In addition, preliminary safety data from an ongoing pediatric clinical trial for sTNFR(p75):Fc indicates that this drug is generally well-tolerated by patients with juvenile rheumatoid arthritis (JRA) (Garrison et al, 1998, Am. College of Rheumatology meeting, Nov. 9, 1998, abstract 584).
As noted above, ENBREL is a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75 kilodalton (p75) TNFR linked to the Fc portion of human IgG1. The Fc component of ENBREL contains the CH2 domain, the CH3 domain and hinge region, but not the CH1 domain of IgG1. ENBREL is produced in a Chinese hamster ovary (CHO) mammalian cell expression system. It consists of 934 amino acids and has an apparent molecular weight of approximately 150 kilodaltons (Smith et al., 1990, Science 248:1019-1023; Mohler et al., 1993, J. Immunol. 151:1548-1561; U.S. Pat. No. 5,395,760 (Immunex Corporation, Seattle, Wash.); U.S. Pat. No. 5,605,690 (Immunex Corporation, Seattle, Wash.).
Approved by the Food and Drug administration (FDA) (Nov. 2, 1998), ENBREL is currently indicated for reduction in signs and symptoms of moderately to severely active rheumatoid arthritis in patients who have had an inadequate response to one or more disease-modifying antirheumatic drugs (DMARDs). ENBREL can be used in combination with methotrexate in patients who do not respond adequately to methotrexate alone. ENBREL is also indicated for reduction in signs and symptoms of moderately to severely active polyarticular-course juvenile rheumatoid arthritis in patients who have had an inadequate response to one or more DMARDs (May 28, 1999). ENBREL is given to RA patients at 25 mg twice weekly as a subcutaneous injection.
Currently, treatments using the sTNFR(p75):Fc (ENBREL, Immunex) preparations, including those described above, are administered subcutaneously twice weekly, which is costly, unpleasant and inconvenient for the patient. xe2x80x9cImportant Drug Warningxe2x80x9d on World Wide Web at fda.gov/medwatch/safety/1999/enbrel.htm; xe2x80x9cNew Warning For Arthritis Drug, ENBRELxe2x80x9d on World Wide Web at fda.gov/bbs/topics/ANSWERS/ANS00954.html; xe2x80x9cENBREL Injections Difficult for Some Patientsxe2x80x9d at dailynews.yahoo.com/h/nm/20000516/hl/arthritis_drugsxe2x80x941.html. Further, relief afforded by this treatment is not sustained. Symptoms associated with an arthritic condition are reduced during treatment with sTNFR(p75):Fc but return upon discontinuation of this therapy, generally within one month. Complications have arisen, including local reactions at the site of injection. Moreover, long-term systemic exposure to this TNF-xcex1 antagonist can impose a risk for increased viral and bacterial infections and possibly cancer. Since this product was first introduced, serious infections, some involving death, have been reported in patients using ENBREL. xe2x80x9cProduct Informationxe2x80x9d on World Wide Web at enbrel.com/patient/html/patpi.htm; xe2x80x9cProven Tolerabilityxe2x80x9d on World Wide Web at enbrel.com/patient/html/patsafety.htm.
Additional relevant references include: U.S. Pat. Nos. 5,858,775; 5,858,355; 5,858,351; 5,846,528; 5,843,742; 5,792,751; 5,786,211; 5,780,447; 5,766,585; 5,633,145; International Patent publications WO 95/16353; WO 94/20517; WO 92/11359; Schwarz, 1998, Keystone Symp., January 23-29, abstract 412; Song et al. (1998) J. Clin. Invest. 101:2615-2621; Ghivizzani et al., 1998, Proc. Natl. Acad. Sci. USA 95:4613-4618; Kang et al., 1997, Biochemical Society Transactions 25:533-537; Robbins et al., 1997, Drug News and Perspect. 10:283-292; Firestein et al., 1997, N. Eng. J. Med. 337:195-197; Muller-Ladner et al., 1997, J. Immunol. 158:3492-3498; and Pelletier et al., 1997, Arthritis Rheum. 40:1012-1019.
There is a need for new, effective forms of treatment for TNF-associated disorders such as RA, particularly treatments that can provide sustained, controlled therapy. The present invention provides compositions and methods for effective and continuous treatment of inflammatory processes of arthritis and other TNF-associated disorders.
All publications and references cited herein are hereby incorporated by reference in their entirety.
The present invention is directed to compositions and methods for reducing TNF levels and/or treatment of TNF-associated disorders of a mammal. The compositions generally comprise a recombinant adeno-associated virus (rAAV) vector that contains a polynucleotide encoding a TNF antagonist. The methods generally employ an rAAV vector to deliver a polynucleotide encoding a TNF antagonist to the mammal, which in turn reduces the levels of TNF and results in palliation of a number of TNF-associated disorders, such as arthritis (including RA), Crohn""s disease, asthma and congestive heart failure. Lowering TNF may in turn reduce levels of other disease causing or contributing agents, such as other inflammatory cytokines. Lowering the levels of soluble TNF in joints exhibiting RA can in turn palliate TNF-associated conditions, such as arthritis, and can reduce an inflammatory response in the joints.
A preferred polynucleotide for the invention in the rAAV vectors described herein is one encoding a tumor necrosis factor receptor (TNFR). Since TNFR is capable of binding to soluble TNF, the introduction of TNFR tends to reduce the levels of TNF in circulation and/or the affected tissues, such as the joint. In some embodiments, the invention provides an rAAV vector comprising a polynucleotide encoding a p75 TNFR polypeptide. In other embodiments, the rAAV vectors of the invention comprise a polynucleotide encoding an Fc (constant domain of an immunoglobulin molecule):p75 fusion polypeptide. In other embodiments, the rAAV vectors of the invention comprise a polynucleotide encoding a fusion polypeptide in which the extracellular domain of TNFR is fused to Fc.
In some embodiments, the rAAV vectors of the invention further comprise a polynucleotide encoding an IL-1 antagonist, such as an IL-1 receptor type II polypeptide.
In another aspect, the invention provides methods for reducing TNF levels in a mammal, which comprise administering (i.e., delivering) any of the rAAV vectors described herein to the mammal in an amount sufficient to reduce TNF levels. In some embodiments, the delivery of an rAAV vector is in an arthritic joint. In some embodiments, these methods further comprise administering a TNF antagonist.
In another aspect, the invention provides methods for reducing an inflammatory response in a mammal, which comprise administering (i.e., delivering) any of the rAAV vectors described herein to the mammal in an amount sufficient to reduce the inflammatory response. In some embodiments, these methods further comprise administering a TNF antagonist.
In another aspect, the invention provides methods for palliating a TNF-associated disoreder, such as an arthritic condition occurring in a mammal, which comprise administering (i.e., delivering) any of the rAAV vectors described herein to the mammal in an amount sufficent to palliate the disorder (such as arthritic condition). In some embodiments, these methods further comprise administering a TNF antagonist.